Taki Masayasu, Wolford Janet L, O'Halloran Thomas V
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
J Am Chem Soc. 2004 Jan 28;126(3):712-3. doi: 10.1021/ja039073j.
Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.
锌和钙是细胞内普遍存在的金属，虽然已经开发了多种用于测量细胞内钙浓度变化的定量探针，但锌的情况并非如此。我们在此描述基于苯并恶唑的比率型锌探针Zinbo-5的设计、合成及特性。这种明亮的荧光报告分子在锌结合形式下的量子产率为0.1，对Zn2+的解离常数在纳摩尔范围内，并且在锌结合后激发和发射最大值都有显著变化。这种细胞可渗透探针的实用性在对哺乳动物细胞的荧光显微镜发射比率成像实验中得到了证明。我们进一步表明，Zinbo-5非常适合双光子激发显微镜比率成像，并且能够轻松揭示单细胞光学平面内细胞内锌浓度的变化。据我们所知，这是双光子激发显微镜应用于锌比率成像的首个实例。这些方法可应用于活细胞中锌生理学的实时发射或激发比率成像研究。
